Neisseria gonorrhoeae metal transporters that subvert nutritional immunity

人类特有的细菌病原体淋病奈瑟菌（Ngo）导致性传播感染 淋病Ngo表达外膜转运蛋白，以TonB依赖的方式获得铁和其他金属。 方式为了防止微生物生长和传染性，人类使用高亲和力金属结合蛋白来 螯合必需金属，这种现象被称为“营养免疫”。Ngo颠覆营养免疫 通过使用专门的TonB依赖性转运蛋白（TdTs），结合并从人体金属中去除金属- 结合蛋白，如转铁蛋白和乳铁蛋白，其螯合铁（Fe）。我们的初步数据显示 两种Ngo外膜转运蛋白TdfH和TdfJ使Ngo能够从Zn- 隔离人类先天免疫蛋白。TdfH使Ngo能够克服锌螯合 由先天免疫蛋白钙卫蛋白（CP）施加，其由嗜中性粒细胞大量产生， 在炎症部位聚集到高浓度。此外，在上一个融资期，我们 确定TdfJ使得能够使用S100 A7（也称为银肩病素）作为唯一的Zn源。另外两 转运蛋白TdfF和TdfG没有很好的定义，但它们的表达是铁抑制的。我们假设 这些金属转运蛋白使Ngo能够克服先天免疫的生长抑制作用， 炎症过程中产生的蛋白质。这些研究意义重大，因为了解Ngo如何获得 人类宿主中的锌和铁等必需金属为开发新疗法提供了新的机会 无论它们是保护性疫苗的靶点，还是关键病毒的小分子抑制剂， 运输机在拟议的研究中要测试的首要假设是，非政府组织颠覆了营养 由嗜中性粒细胞产生的宿主金属螯合蛋白（包括S100蛋白）产生的免疫力 和上皮细胞，通过部署外膜转运蛋白，其结合并释放这些蛋白质 他们被隔离的过渡金属货物为了检验这些假设，提出了以下具体目标： 目的1将询问Ngo在与人类宫颈细胞相互作用时所感知的金属环境， 原代人中性粒细胞，在人类感染中重要的细胞类型。我们将在以下情况下确定感染情况 其中Ngo使用荧光报告子和RNA-seq方法经历Fe和Zn限制。目标2将 采用结构生物学方法，确定Ngo TdTs破坏宿主营养免疫的机制 和蛋白质-蛋白质相互作用技术。目的3将阐明抑制生物学效应的后果。 TdTs在体外和与宿主细胞的功能。这些研究将测试单克隆抗体，TdT诱变 和S100蛋白抑制剂对宿主细胞存活和金属吸收的影响。总的来说，这些创新的研究 将建立在前一个资助期令人兴奋的发现的基础上，并将进入创新的新领域， 进行调查部署最先进的技术拟议的研究将揭示新的基本光 Ngo用来克服人类宿主所施加的营养免疫的致病机制。

The human-specific bacterial pathogen Neisseria gonorrhoeae (Ngo) causes the sexually-transmitted infection gonorrhea. Ngo expresses outer membrane transporters to acquire iron and other metals in a TonB-dependent manner. In order to prevent microbial growth and infectivity, humans use high affinity metal-binding proteins to sequester essential metals, a phenomenon known as “nutritional immunity.” Ngo subverts nutritional immunity by using dedicated TonB-dependent transporters (TdTs) that bind to and remove the metals from human metal- binding proteins such as transferrin and lactoferrin, which sequester iron (Fe). Our preliminary data indicates that two Ngo outer membrane transporters, TdfH and TdfJ, enable Ngo to internalize zinc (Zn) from Zn- sequestering, human innate immunity proteins. TdfH affords Ngo the ability to overcome Zn sequestration imposed by the innate immunity protein calprotectin (CP), which is produced in abundance by neutrophils and accumulates to high concentrations at sites of inflammation. Furthermore, in the prior funding period, we determined that TdfJ enables the use of S100A7, also known as psoriasin, as the sole Zn source. Two other transporters, TdfF and TdfG, are not as well defined, but their expression is iron-repressed. We hypothesize that these metal transporters enable Ngo to overcome the growth inhibitory effects of the innate immunity proteins produced during inflammation. These studies are significant because knowing how Ngo acquires essential metals like Zn and Fe from the human host opens new opportunities for developing new therapeutics against ‘superbug’ strains, whether they be targets for a protective vaccine or small molecule inhibitors of crucial transporters. The overarching hypothesis to be tested in the proposed study is that Ngo subverts nutritional immunity imposed by host metal-chelating proteins including S100 proteins, which are produced by neutrophils and epithelial cells, by the deployment of outer membrane transporters, which bind to and relieve these proteins of their sequestered transition metal cargo. To test these hypotheses, the following specific aims are proposed: Aim 1 will interrogate the metal environment sensed by Ngo when interacting with human cervical cells and primary human PMNs, cell types important in human infection. We will identify the infection conditions under which Ngo experiences Fe and Zn limitation using fluorescent reporters and RNA-seq approaches. Aim 2 will define the mechanisms for subversion of host nutritional immunity by Ngo TdTs, employing structural biology and protein-protein interaction techniques. Aim 3 will elucidate the consequences of inhibiting the biological functions of the TdTs in vitro and with host cells. These studies will test monoclonal antibodies, TdT mutagenesis and S100 protein inhibitors for impacts on host cell survival and metal uptake. Overall, these innovative studies will build on exciting findings from the prior funding period and will also move into innovative new areas of investigation, deploying state-of-the-art technologies. The proposed studies will shed new light on fundamental pathogenic mechanisms used by Ngo to overcome nutritional immunity imposed by the human host.